Cerebral small vessel disease (CSVD), a recognized priority established by the NINDS, is a major cause of cognitive impairment. In this proposal, we present the novel idea that antigens derived from the gut microbiota constitute a fundamental source for the inflammation underlying CSVD. We propose that gut dysbiosis allows bacteria and bacterial components to translocate the gut epithelial barrier and ultimately gain access to the brain where they initiate and maintain inflammation necessary for the development of CSVD. The hypothesis is supported with strong preliminary data: (1) gut dysbiosis occurs in spontaneously hypertensive stroke-prone rats (SHRSP), a relevant model for CSVD, compared to its parent strain, WKY rats; (2) altering the gut microbiome in WKY rats to resemble that of SHRSP is accompanied by pathological changes occurring in SHRSP. Altering the gut microbiome in SHRSP rats to resemble that of WKY rats attenuates these pathological changes; (3) brains of SHRSP contain 52% more bacterial DNA than WKY rats; (4) gram-negative bacteria represent a substantially greater proportion of the overall bacterial DNA in brains of SHRSP compared to WKY rats; and (5) lipopolysaccharides (LPS), bacterial endotoxin derived from gram negative bacteria, are substantially increased in brains of SHRSP compared to WKY rats. In Specific Aim 1 we will determine if CSVD can be produced or prevented through manipulation of the gut microbiota. If gut dysbiosis is an underlying cause of CSVD then we should be able to induce CSVD in control WKY rats, the parent strain for the SHRSP, by inducing dysbiosis. Alternatively, we should be able to abolish or delay the onset of CSVD in SHRSP rats by preventing dysbiosis. In Specific Aim 2 we will identify gut and brain bacteria and bacterial components involved with the initiation of CSVD. (a) We will analyze the bacterial DNA composition of the gut and brain by sequencing the bacterial 16s rRNA gene from feces, cecal content, and brain. Using targeted qPCR, we will further identify bacteria to the species level. (b) We will determine if intact bacteria are resident in the brain using RT-PCR and fluorescence in situ hybridization of SHRSP and WKY rats. In Specific Aim 3 we propose to track the movement of bacteria and bacterial components, a process termed translocation, from the gut to the brain. First, we will determine if fluorescently labeled LPS and peptidoglycan track to the brain when gavaged into the gut of SHRSP and WKY rats. Second, we will employ bacteria that we have engineered with reporters to track bacteria from gut to brain and determine if gram- negative bacteria are capable of translocating from gut to brain more efficiently in hosts developing CSVD. If our hypothesis is valid, then the gastrointestinal tract (GI) can affect the health of the brain. Establishing the gut microbiome as a source for inflammation related to CSVD could dramatically refocus our attention on the GI tract as a potential cause for brain pathologies, as well as provide a target for therapeutic intervention.